FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.


Fig. S1. Additional analysis of CRISPR screen performed in Raji and Namalwa cells, related to
(A) Horizontal scatter plot of DrugZ-generated ranking of the Raji mafosfamide CRISPR screen. NormZ values are plotted on the Y-axis, and gene names are plotted on the X-axis. (B) Gene Set Enrichment Analysis (GSEA) analysis of the score obtained in the Namalwa screen for ICL repair and HR. (C) Network analysis displaying protein physical interactions for sensitizer genes common to Namalwa and Raji CRISPR screens using Cytoscape and the GeneMANIA package. (D) Representative scatter plots of BFP or mCherry-positive RPE1-hTERT p53-/-cells analyzed by flow cytometry and presented in Figure 1G. Fig.2 (A) WCE of U2OS cells depleted or not for FIRRM were analyzed by immunoblot with an anti-FIRRM antibody. Anti-a-Tubulin was used as a loading control. (B) Representative images of U2OS cells treated with a siFIRRM or non-targeting siRNA for 48 hrs. Cells were processed for 53BP1 and CCNA1 (Cyclin A) immunofluorescence. Scale bar = 5 μm.

Fig. S2. Validation of FIRRM knockdowns and knockouts, analysis of 53BP1 nuclear bodies and additional senescence assays, related to
(C) Quantification of 53BP1 nuclear bodies per CCNA1-negative cell as shown in (B). Data are represented as the mean ± SD (n = 3 independent experiments. A minimum of 100 cells were analyzed per condition per experiment). (D) Representative images of data shown in Fig.2C. (E) WCE of RPE1-hTERT WT or p53-/-cells depleted or not for FIRRM with the indicated shRNA were analyzed by immunoblot with an anti-FIRRM antibody. Anti-b-actin was used as a loading control. (F) RT-qPCR for FIRRM was performed on IMR90 cells. Expression was normalized against GADPH and reported as relative to shCtrl. Data are represented as the mean ± SD (n = 3 independent experiments). (G). Quantification of IMR90 cells depleted or not for FIRRM and stained with SA βgalactosidase. Data are represented as the mean ± SEM (n = 2 independent experiments with 3 technical replicates). (H) Representative images of IMR90 cells depleted or not for FIRRM and stained with SA βgalactosidase. Scale bar = 100 µm (I) RT-qPCR for CDKN1A (p21), MDM2 and FGF2 was performed on IMR90 cells. Expression was normalized against GAPDH and reported as relative to shCtrl. Data are represented as the mean ± SD (n = 3 independent experiments). (J) WCE of RPE1-hTERT p53-/-clonal cells depleted or not for FIRRM with the indicated sgRNA were analyzed by immunoblot with an anti-FIRRM antibody. Anti-b-actin was used as a loading control. (K) Karyotype representation of RPE1-hTERT p53-/-cells depleted (sgFIRRM-1) for FIRRM displaying aberrant chromosome copy numbers through metaphase analysis. (L) Representative scatter plots of PI and/or Annexin V-positive RPE1-hTERT p53-/-cells analyzed by flow cytometry and presented in Fig.2I. Data were analyzed with an unpaired t-test with Welch's correction (panel C) or an ordinary oneway ANOVA test with Dunnett's multiple comparison test (panels F, G and I). *p<0.05, ****p<0.0001

Fig. S3. Characterization of FIRRM knockdown and knockout cells related to Fig 3 (A)
Quantification of RPE1-hTERT p53-/-cells survival in response to ICL-inducing agent. Cells were transduced with lentiviral particles containing sgRNAs against FIRRM, FANCA or a non-targeting control (sgCtrl). After selection with puromycin, cells were seeded in 96-well plates and treated with mafosfamide (MAF) or cisplatin (Cis.). Both drugs were added at a maximum concentration of 50 µM in a two-fold serial dilution until 0.097 µM. Data are represented as the mean ± SEM (n = 5 independent experiments). (B) Representative images of U2OS 2-6-5 cells depleted or not for FIRRM. Cells were treated as in Figure 3E and processed for g-H2AX (left) and 53BP1 (right) immunofluorescence. Scale bar = 5 μm. (C) WCE of U2OS 2-6-5 cells depleted or not for FIRRM were analyzed by immunoblot with an anti-FIRRM antibody. Anti-a-Tubulin was used as a loading control. (I) Quantification of cell cycle distribution in U2OS cells depleted or not for FIRRM, FIGNL1 or both. Cells were fixed and processed for EdU analysis through flow cytometry. Data are represented as the mean ± SD (n = 3 independent experiments, at least 30,000 cells were analyzed per experiment in each condition). (J) Representative images of U2OS 2-6-5 cells depleted or not for FIRRM. Cells were treated as in Figure 3K and processed for g-H2AX (left) and 53BP1 (right) immunofluorescence. Scale bar = 5 μm. (K) Quantification of the indicated protein with the empty mCherry-LacRnls construct. U2OS 2-6-5 cells were treated as in Figure 3K. Data are represented as the mean ± SD (n = at least 5 independent experiments, 50 foci quantified per experiment in each condition). (L) Chemogenomic profiling using the NormZ-score from the CRISPR screens performed in RPE1 hTERT Cas9 p53-/-cells with the indicated DNA damaging agents as detailed in (38). (M) Viability assay in cells treated with DNA damage-inducing agent. U2OS cells were treated with the indicated siRNA. Forty-eight hours later, cells were either exposed to 2 µM cisplatin (Cis.), 150 µM formaldehyde (FormA), 150 nM mitomycin C (MMC), 3 µM BMN-673 for 48 hrs, to 2 hrs of 4 mM hydroxyurea (HU) followed by 46 hrs recovery, to 24 hrs of 1 mM HU followed by 24 hrs recovery, to 10 J/m2 UV or 10 Gy X-rays followed by 48 hrs recovery. Cells were counted with a hemacytometer and normalized against the number of cells quantified in the untreated condition. Data are represented as the mean ± SD (n = 4 independent experiments).
Data were analyzed using a one-way Welch's ANOVA test with Dunnett's multiple comparison test (panels A, F, G and H) or with an unpaired t-test with Welch's correction (panels K and M). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001  Fig.4C were analyzed by immunobloting with anti-FIRRM and anti-RAD51 antibodies. Anti-a-tubulin antibody was used as a loading control. (D) Schematic representation of the biotinylation of FIRRM proximal endogenous proteins using TurboID technology. Cutoff for proximity tagging is reported at 10nm. (E) WCE extracts of HEK293T Flp-InTM cells induced or not for mTurboID expression with 1µM tetracycline and supplemented or not for 50 µM biotin were analyzed by immunoblot with anti-streptavidin and anti-Flag antibodies. Anti-a-tubulin was used as a loading control. (F-G) WCE extracts of U2OS 2-6-5 cells transfected with the indicated GFP-(F) or mCherry-LacRnls-(G) proteins were analyzed by immunoblot with anti-GFP or anti-mCherry antibodies. GFP alone was used as control (Ctrl). Anti-a-tubulin was used as a loading control.
(H) Quantification of the ratio of indicated protein foci intensity over ER-mCherry-LacR-FOK1-DD foci intensity as shown in (I). Data are represented as the mean ± SD (n = 4 independent experiments, 50 foci quantified per experiment in each condition). (I) U2OS 2-6-5 cells were depleted or not for FIGNL1 (for 48 hrs and ER-mCherry-LacR-FOK1-DD expression was induced for 4 hrs before fixation. Cells were then processed for g-H2AX, 53BP1, BRCA1, BRCA2 or RAD51 immunofluorescence. Scale bar = 5 μm. (J) Quantification of GFP-positive HeLa DR-GFP cells depleted or not for RAD51, FIGNL1 and/or FIRRM. HeLa cells containing a DR-GFP reporter cassette were transfected and analyzed as described in Figure S3H Data were analyzed with an unpaired t-test with Welch's correction (panels B, H, K and L) or using a one-way Welch's ANOVA test with Dunnett's multiple comparison test (panel J). *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001 Fig. 5. (A) WCE of U2OS 2-6-5 cells transfected with the indicated mCherry-LacRnls-FIGNL1 constructs were analyzed by immunoblot with an anti-mCherry antibody. Anti-a-tubulin antibody was used as a loading control. KDm: K447A / D500A double mutant. (B) Quantification of mCherry-LacRnls constructs colocalizing with GFP-FIRRM (left) or RAD51 (right) in U2OS 2-6-5 cells. Cells were treated and processed as described in Figure 4C. Data are represented as the mean ± SD (n = 3 independent experiments, 50 cells quantified per experiment in each condition). (C-D) Representative images of data from (B). U2OS 2-6-5 cells were transfected with indicated mCherry-LacR construct and then fixed, processed for RAD51 immunofluorescence (D), and mounted for confocal analyses. Scale bar = 5 μm. (E) U2OS 2-6-5 cells were transfected with the indicated GFP-FIRRM constructs and analyzed by immunoblot with a GFP antibody. GFP was used as control. Anti-a-tubulin antibody was used as a loading control. (F) Quantification of cell survival in response to cisplatin in GFP-FIRRM WT and GFP-FIRRM ∆WCF U2OS. Cells were treated with a Ctrl siRNA and supplemented or not with 1 µg/mL Dox prior to treatment with 1 µM cisplatin. Cells were then processed as in Figure Table S1. NormZ values for Namalwa and Raji CRISPR screens. Provided as an Excel file.